There are two general methods for the refrigerated storage of human red blood cells: 1) refrigerated storage in the original anticoagulant solution; 2) refrigerated storage after separation of the red cells from the anticoagulant solution and the plasma, and resuspension of the cells in a solution that is specifically designed for red cell storage.
1) For storage in the original anticoagulant, whole blood is conventionally drawn into a solution containing citrate, phosphate, dextrose (d-glucose) and adenine (CPDA-1) at pH 5.7. The blood is centrifuged at about 1500 g (soft spin) and the plasma removed leaving a red cell suspension with an hematocrit of about 75%. Platelets can be removed from the plasma by a second sedimentation.
2) For resuspension of the red cells and storage in a preservation solution, blood is conventionally drawn into a solution containing only citrate, phosphate and glucose at pH 5.7. The blood is centrifuged at the same speed as described in (1) above but the red cells are then resuspended in either Adsol of Nutricel (see Table at pH 5.7 and 5.8 respectively, resulting in a red cell suspension at an hematocrit of approximately 55%.
During storage, human red blood cells undergo morphological and biochemical changes, including decreases in the cellular levels of adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG), changes in cellular morphology and progressive hemolysis. The concentration of ATP, after a brief initial rise, progressively declines to between 30 and 40% of its initial level after six weeks of storage. The fluidity of the cell membrane of red cells, which is essential for the passage of red cells through the narrow channels in the spleen and liver, is loosely correlated with the level of ATP. The level of 2,3 DPG falls rapidly after about 3 or 4 days of storage and approaches zero by about 10 days. 2,3 DPG is associated with the ability of the hemoglobin in the red cells to deliver oxygen to the tissues. Morphological changes occur during storage, ultimately leading to the development of spicules on the cells (echinocytosis). These spicules can bud off as vesicles, radically changing the surface-to-volume ratio of the cells and their ability to deform on passing through narrow channels. Such cells will be filtered out of the circulation by the spleen and liver following transfusion. To be acceptable for transfusion at least 75% of the red cells that are transfused must be circulating 24 hours following the transfusion. Shelf life of red blood cells is determined on this basis. The concentration of ATP and the morphology of red cells serve as indicators of the suitability of stored cells for transfusion.
In order to prolong the shelf life of transfusible red blood cells it is necessary to store the cells or treat them in some manner that prevents a rapid decline in ATP and, if possible, 2,3 DPG (see e.g., Harmening, U.S. Pat. No 4,112,070 and Goldstein, U.S. Pat. No. 4,427,777). Solutions that prolong the shelf life of red cells are known (see e.g., Harmening, supra. and Meryman, U.S. Pat. No. 4,585,735, which disclosure is herein incorporated in its entirety by reference thereto). Typically such solutions contain citrate, phosphate, glucose and adenine and occasionally other ingredients that function to prolong shelf life by maintaining the level of ATP in the cells. Minakami et al. ((1975) In: Brewer, C. J., ed. Erythrocyte Structure and Function, New York, Liss, pp. 149-166) report that glycolytic activity is enhanced in red blood cells if the intracellular pH (hereinafter pH.sub.i) measured at 4.degree. C. is about 7.4 and suggest that pH.sub.i is a parameter that should be considered with respect to blood preservation. Solutions that maintain high levels of both ATP and 2,3 DPG, during long term storage without artificial intervention (see, e.g. Harmening, supra.) or without the inclusion of compounds such as ammonium, not licensed for transfusion (see, e.g., Meryman, supra.), are not, however, known.
Procedures and solutions have been devised that permit some of the declines in ATP and 2,3 DPG and the morphological changes associated with long-term storage to be reversed and thereby rejuvenate the red blood cells. Rejuvenating solutions, however, are not suitable for transfusion; they must be removed prior to transfusing the cells. There is, thus, a risk of contamination associated with this procedure. Federal law requires that cells that have been so-treated must be transfused within 24 hours in order to minimize the risk of bacterial growth. Devices have now been developed that permit removal of the rejuvenation solution in a closed system without subjecting the cells to the risk of contamination. However, after rejuvenation the cells must be washed with a solution that is suitable for transfusion. Conventional wash solutions, such as glucose-saline solutions, are not, however, suitable for storage beyond 24 hours.
There are other instances in which red blood cells must be washed. For example, cells that are stored by freezing in glycerol must be deglycerolized by washing prior to use. Moore et al. (1987, Vox Sang. 53:19-22) have reported deglycerolizing frozen red cells using a phosphate-buffered sodium chloride wash solution with resuspension in a solution containing adenine, ascorbate-2-phosphate, trisodium phosphate, dextrose and mannitol at a pH of 11.0 and an osmolality of 446 mOsm. Both ATP and 2,3 DPG were adequately maintained for 21 days. However, ascorbate-2-phosphate has not been licensed for use in a solution for transfusion. In a subsequent publication, Carmen et al. (1988, Transfusion 28:157-161) reported that red cells stored for only 5 weeks in a solution containing ascorbate-2-phosphate lost ATP to a level of 22.2% of initial value with 24-hour survival falling below 75%.
Red blood cells that have been subjected to other treatments must also be washed prior to transfusion. For example, Goldstein, supra., discloses a method for converting type B red cells into type O cells by removing the terminal galactose moiety of the B-antigenic determinant of stroma from type A cells under conditions wherein the cells do not lose their cellular functions so that they are suitable for transfusion. The enzymatic cleaving of the terminal galactose must be performed at low pH. Following enzymatic treatment the red cells are washed with isotonic sodium chloride that is buffered with 0.01M potassium phosphate buffer at pH 7.4 in part to wash out residual enzyme and in part to raise the pH. Cellular metabolic studies indicate that ATP levels remain above 90% and 2,3 DPG levels are 80-90% immediately after this treatment, but these levels would not be maintained during subsequent storage in this washing solution.
Transfusion of red blood cells poses a risk of viral infection in a recipient from blood that has been obtained from donors that are infected with viruses, such as non A non B hepatitis virus and human immunodeficiency virus. In order to mitigate this risk procedures have been reported whereby the cells are treated with agents that inactivate the viruses. Red cells that are detoxified, however, must then be washed in order to remove the inactivating agent in order to render them suitable for transfusion. No resuspension solution is available that will permit subsequent storage of such cells.
In certain circumstances it is desirable to extend the shelf-life of refrigerated red cells beyond the current 42 days. Autologous units drawn for use in elective surgery may expire before the surgery can be performed. It has also been proposed that blood be stored for several months to permit retesting the donor for evidence of AIDS or hepatitis infection. Other than by freezing, which is labor intensive and expensive, no such capability is known to exist.
Because of the critical need for transfusible red blood cells, it is of great importance to develop methods and solutions that not only maintain high intracellular levels of both ATP and 2,3 DPG, good morphology and low hemolysis after washing but also to develop methods for the routine collection and resuspension of unwashed red cells with better storage characteristics than are achieved by current procedures. Further there is a need to develop solutions that are suitable for both washing and storing transfusible red blood cells.
A great need in the art is to develop procedures for storing red blood cells after collection, but without washing; such a method would have substantial clinical importance.
It is also desirable that the quantity of adenine in transfusable red cells be reduced or eliminated because of concern regarding their nephrotoxicity.